The invention is directed to a method for determining the concentration of asymmetric dimethylarginine simultaneously with arginine and symmetric dimethylarginine (SDMA) in biological samples by HPLC-MS-MS.
In biological systems, the amino acid arginine represents, inter alia, a starting material for the synthesis of the messenger nitric oxide (NO) by the enzymes of the group of nitrogen monoxygenases (NOS). Sufficient availability of this messenger is also a prerequisite for the physiological function of the endothelial cells in the cardiovascular system, whereas a deficiency in NO is related to endothelial dysfunction and the associated disease states, such as arteriosclerosis, hypertension or stroke.
Arginine integrated in proteins is metabolized in the body on a further metabolic pathway via the enzyme group of the protein-methyl-transferases (PRMT) into monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). These amino acids are released during enzymatic protein decomposition. The released methylated arginine derivatives MMA and ADMA inhibit the function of NOS and thereby reduce the production of NO. MMA plays hereby only a minor role, because its effective concentration attains only approximately 10% of that of ADMA. Although SDMA itself does not inhibit NOS, it shares with ADMA the transport mechanism through the cell membranes and can therefore indirectly affect the generation rate of NO.
Elevated plasma levels of ADMA are described in a number of disease states, such as hypertension, hypercholesterolemy, kidney failure or diabetes mellitus, and it can be safely assumed that an elevated plasma level of ADMA is a direct risk factor for cardiovascular diseases.
For investigating these diseases and therapeutic approaches, a steadily increasing demand for the determination of ADMA in different biological fluids, such as blood plasma, urine or a cell culture medium, can be expected.
ADMA is excreted in biological systems both through the kidney in unchanged form or enzymatically divided into citrulline and dimethylamine. This means that ADMA and SDMA increase in the blood in patients with kidney failure. A kidney transplant normalizes the SDMA level, but lowers the ADMA level only slightly, because dimethylamine-dimethylamino hydrolase (DDAH) becomes the main metabolization pathway for ADMA (SDMA is not metabolized). Two isoforms exist: DDAH I has been found in tissue with neuronal NOS, whereas DDAH II is found in tissue with endothelial NOS. A number of pathological stimuli increase oxidative stress, such as oxidized LDL-cholesterol, cytokine, hyperhomocysteinemy, or hyperglycomy. Each of these stimuli cause a decrease in the DDAH activity due to increased formation of oxygen radicals and hence an increase in the ADMA concentration, as depicted in the Figure illustrating the prior art. This effect can be compensated in vitro through administration of antioxidants, restoring the DDAH activity.
The activity of DDAH is of considerable importance for the equilibrium concentration of ADMA in biological systems. It is known that the activity of DDAH is affected by external factors, such as oxidative stress. A decreased activity causes increased ADMA levels with their negative consequences.
The quantitative measurement of ADMA in biological samples is made more difficult because, in addition to ADMA, a large number of other constituents are present in these samples, wherein the other physiological amino acids and in particular SDMA which has a structure similar to that of ADMA make a quantitative determination difficult. A number of methods for determining ADMA in biological samples are generally known from the published literature. Most widely used is the method of high pressure-liquid chromatography with fluorescence detection. Arginine, ADMA, SDMA, optionally MMA and an internal standard, are hereby extracted from biological samples by ion exchange-solid phase extraction (SPE), the extract is reacted with orthophthalic aldehyde and a mercaptan (e.g., 2-mercaoptoethanol, 3-mercaptopropionic acid) to form a fluorescing derivate; the derivates of the aforementioned compounds are separated with HPLC and quantitatively measured using fluorescence detection. A large number of modifications of this method have been described (see, for example, Chen B M, Xia L W, Zhao R Q. Determination of N(G),N(G)-dimethylarginine in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1997; 692: 467-71, Meyer J, Richter N, Hecker M. High-performance liquid chromatographic determination of nitric oxide synthase-related arginine derivatives in vitro and in vivo. Anal Biochem 1997; 247: 11-6, Pettersson A, Uggla L, Backman V. Determination of dimethylated arginines in human plasma by high-performance liquid chromatography, J Chromatogr B Biomed Sci Appl 1997; 692: 257-62, Pi J, Kumagai Y, Sun G, Shimojo N. Improved method for simultaneous determination of L-arginine and its mono- and dimethylated metabolites in biological samples by high-performance liquid chromatography, J Chromatogr B Biomed Sci Appl 2000; 742: 199-203, Dobashi Y, Santa T, Nakagomi K, Imai K. An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction. Analyst 2002; 127: 54-9, Teerlink T, Nijveldt R J, de Jong S, van Leeuwen PAM. Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography. Anal Biochem 2002; 303:131-7, Marra M, Bonfigli A R, Testa R, Testa I, Gambini A, Coppa G. High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde. Anal Biochem 2003; 318: 13-7 Heresztyn T, Worthley M I, Horowitz J D. Determination of 1-arginine and N(G), N(G)- and N(G), N(G′)-dimethyl-1-arginine in plasma by liquid chromatography as AccQ-Fluor™ fluorescent derivatives. J Chromatogr B AnalytTechnol Biomed Life Sci 2004; 805: 325-9 and Zhang W Z, Kaye D M. Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization. Anal Biochem 2004; 326: 87-92). A major disadvantage of this method is the extraction with SPE, which is labor intensive, expensive and error prone. Other chromatographic separation methods are based on capillary electrophoresis with fluorescence detection (see, for example, Causse E, Siri N, Arnal J F, Bayle C, Malatray P, Valdiguie P et al. Determination of asymmetrical dimethylarginine by capillary electrophoresis-laser-induced fluorescence. J Chromatogr B Biomed Sci Appl 2000; 741: 77-83, und Trapp G, Sydow K, Dulay M T, Chou T, Cooke J P, Zare R N. Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl-L-arginine and structurally related amino acids: quantitation in human plasma. J Sep Sci 2004; 27: 1483-90), or gas chromatography with mass-spectrometric detection after a corresponding extraction and derivatization of ADMA and optionally arginine and SDMA (see, for example, Tsikas D, Schubert B, Gutzki F M, Sandmann J, Frolich J C. Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative. J Chromatogr B AnalytTechnol Biomed Life Sci 2003; 798: 87-99 and Albsmeier J, Schwedhelm E, Schulze F, Kastner M, Boger R H. Determination of N(G),N(G)-dimethyl-I-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry. J Chromatogr B AnalytTechnol Biomed Life Sci 2004; 809: 59-65). Newer methods applying the HPLC-MS technology have also been described (see Huang L F, Guo F Q, Liang Y Z, Li B Y, Cheng B M (2004) Simultaneous determination of L-arginine and its mono- and dimethylated metabolites in human plasma by high-performance liquid chromatography-mass spectrometry. Anal Bioanal Chem 380: 643-649, Martens-Lobenhoffer J, Krug O, Bode-Boger S M (2004) Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique. J Mass Spectrom 39:1287-1294; Kirchherr H, Kuhn-Velten W N (2005) HPLC-tandem mass spectrometric method for rapid quantification of dimethylarginines in human plasma. Clin Chem 51:249-252, and Schwedhelm E, Tan-Andresen J, Maas R, Riederer U, Schulze F, Boger R H (2005) Liquid chromatography-tandem mass spectrometry method for the analysis of asymmetric dimethylarginine in human plasma. Clin Chem 51:1268-1271). These methods partially require derivatization of the analytes prior to chromatography or require complete chromatographic separation of ADMA and SDMA for obtaining reliable quantitative results. A basically different approach for determining of ADMA in biological samples is the determination through immunological reactions using specific antibodies. Commercially available reagent kits operate according to the ELISA process (enzyme-linked immunosorbent assay). However, this process is in principle only capable of quantifying a single analyte, in this case ADMA. Unlike with chromatographic methods, a simultaneous determination of, for example arginine or SDMA, is not possible.